Wir sind besonders dankbar, dass Seth Darst für die Bereitstellung von RNAP und MFD Proteine ​​und Expressionsvektoren. Wir sind auch danken Sergei Borukhov für die Bereitstellung von Greb und Dan Zhang für den technischen Support. Diese Arbeit wurde durch ein Stipendium der National Institutes of Health (MOD) und durch ein Marie-Josée und Henry Kravis Fellowship an der Rockefeller University (RTP) unterstützt.

Diese Methode wurde in der Forschung in gemeldet verwendet <a href="http://www.sciencemag.org/cgi/content-nw/full/327/5965/590/F3">Pomerantz und O&#39;Donnell, Science 327, 590-592 (2010)</a> . 1. Montage eines gestoppt RNAP Dehnung Komplex wurde wie folgt durchgeführt: 500 nM Endkonzentration von E. coli RNAP σ 70 hat er wurde mit 5 nM Endkonzentration von einem biotinylierten 3,6 kb DNA-Template enthalten, die T7A1 Promoter in 100 ul Puffer A (20 mM Tris-Cl (pH 7,5), 8 mM MgCl 2, 0,5 mM EDTA, 5 gemischt mM DTT, 10% Glycerin) für 10 min bei 37 ° C. 100 uM der APU und 40 uM jedes GTP, ATP und CTP wurden für weitere 10 min bei 37 ° C aufgenommen 2. Immobilisierung des gestoppt RNAP Dehnung Komplexes auf Streptavidinbeads wurde wie folgt durchgeführt: 200 ul Streptavidin-Magnetpartikel Kügelchen (Invitrogen) wurden, um die Reaktion über 10 min bei Raumtemperatur aufgenommen. 3. Die Reinigung der immobilisierten gestoppt RNAP Dehnung Komplex wurde wie folgt durchgeführt: Die Perlen (von oben) wurden 5 mal mit 0,9 ml Puffer A mit 0,75 M NaCl, 200 ug / ml Heparin und 20 pg / ml ssDNA (Der Überstand wurde nach jedem Waschschritt durch magnetische Separation entfernt.) Anschließend die gewaschenen Perlen wurden 2 mal mit 0,9 ml Puffer A gewaschen 4. Die Bildung einer Replikationsgabel stromabwärts bezüglich der Kopf-an RNAP wurde wie folgt durchgeführt: Die Perlen (von oben) wurden in 100 ul New England Biolabs Puffer 4 und 10 Einheiten von Sap I (New England Biolabs) resuspendiert wurde für 10 min bei 37 ° C. Die Perlen wurden 3 mal mit 0,9 ml Puffer A gewaschen und dann in 50 ul von Quick T4 Ligationsreaktion Puffer (New England Biolabs) resuspendiert. 2 ul von Quick T4-Ligase (New England Biolabs) wurde zusammen mit 6 nM Endkonzentration von pre-geglüht gespaltene DNA (RP10, RP22, RP25) für 10 min bei Raumtemperatur aufgenommen. Die Perlen wurden 3 mal mit 0,9 ml Puffer A gewaschen 5. Versammlung der replisome und Initiierung von führenden Strang-Synthese an der Replikationsgabel wurde wie folgt durchgeführt: 448 pmol der DnaB (als Hexamer) wurde mit den Beads 150 ul Puffer A (20 mM Tris-Cl (pH 7,5), 8 mM MgCl 2, 0,5 mM EDTA, 5 mM DTT, 10% Glycerin) für 30 s bei 23 ° C. 4,9 pmol des Pol III * (Pol III HE minus β), 15 pmol von β, 2 mM ATP und 60 uM dGTP und dATP wurden auf ein Volumen von 200 ul zugegeben und weitere 5 min bei 37 ° C. Replication wurde nach Zugabe von 10 pg SSB und 24 uM dATP und dTTP mit initiiert [α-32 P] dTTP und [α-32 P] dCTP (spezifische Aktivität, 3000-5000 cpm / pmol) auf ein Endvolumen von 250 ul. Die Reaktionen wurden nach 10 min nach Zugabe von 12 ul 500 mM EDTA beendet. 6. Reinigung und Konzentrierung der radioaktiv markierten DNA-Produkte wurde wie folgt durchgeführt: Die Perlen (von oben) gekocht wurden nach der Reaktion wurde beendet, um die DNA aus den Perlen zu entfernen. Ein verbleibender DNA übrigen an der Perlen wurde durch die Behandlung der Perlen mit Proteinase K in einem Volumen von 10 ul für 30 min bei 50 ° C entfernt Die Perlen wurden dann gekocht und der Überstand wurde durch magnetische Separation entfernt. Der kombinierte Überstand, der die DNA wurde unter Verwendung des Qiagen PCR Cleanup Kit. Gereinigtes radioaktiv markierten DNA-Produkte wurden dann in einem alkalischen Agarosegel analysiert. * Replication in der Abwesenheit von einem Kopf-an RNAP gestoppt Dehnung komplex wie oben ausgeführt wurde, aber σ 70 wurde weggelassen. Repräsentative Ergebnisse: Collision der replisome mit einem Kopf-an RNAP führt in der Regel zwei Produkten von 2,5 kb und 3,6 kb Länge. Die 2,5 kb Produkt ist die Länge der DNA aus der Gabel, die gestoppt RNAP und Ergebnisse aus replisome Abwürgen beim Zusammenstoß mit der RNAP. Das 3,6 kb Produkt stellen in voller Länge DNA und Ergebnisse aus entweder unvollständig Belegung des Promotors durch RNAP oder teilweise replisome Durchlesen durch den Kopf-an RNAP. Ein gutes oder genaues Ergebnis zeigt, dass ca. 50% in voller Länge DNA hergestellt wird. Doch in einigen Fällen weniger Belegung des Promotors durch RNAP auftritt und einen höheren Prozentsatz von Volllängen-DNA ist, da eine größere Population von replisomes beobachtet nicht begegnen einem Kopf-an RNAP. Replikation in Abwesenheit eines gestoppt RNAP Ergebnisse in nur voller Länge DNA (3,6 kb). <img src="/files/ftp_upload/1919/1919fig1.jpg" alt="Abbildung 1" /> Abbildung. 1 Die replisome wird von einem Kopf-an RNAP behindert. (A) Versuchsaufbau. A E. coli RNAP gestoppt Dehnung Komplex auf einem biotinylierten DNA mit den T7A1 Promoter wie zuvor beschrieben (9) montiert wurde. Kurz gesagt wurde ein Stillstand Dehnung Komplex 20 bps stromabwärts vom Promotor durch Inkubation RNAP-Holoenzym mit biotinylierten DNA für 10 min durch Zugabe von APU, GTP, ATP und CTP für weitere 10 min, gefolgt gebildet. Streptavidin Beads wurden für weitere 10 min dann die Kügelchen wurden fünfmal mit je 0,9 ml Puffer mit 0,75 M NaCl, 200 mg / ml Heparin und 100 nM Einzelstrang-DNA zu instabilen RNAP-DNA-Komplexe und überschüssige NTPs entfernen hinzugefügt . Die DNA wurde dann mit SAPI verdaut, gewaschen und ligiert, um ein Pre-geglüht gespaltene DNA. Die Perlen wurden erneut vor der Montage des replisome gewaschen. Der Veranstalter ist 2,5 kb aus der Gabel liegt und orientiert sich an die Transkription auf die Gabel. (B) DnaB wurde hinzugefügt, dann replisome montiert wurde und die Replikation wurde entweder in der Gegenwart begonnen werden (Spur 2) oder Abwesenheit (Spur 1) eines Kopf-an RNAP.

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D., Michel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+fork+collapse+at+replication+terminator+sequences.">Replication fork collapse at replication terminator sequences.</a> Embo J. 21, 3898-3898 (2002).</li><li>Pomerantz, R. T., O'Donnell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replisome+mechanics:+insights+into+a+twin+DNA+polymerase+machine.">Replisome mechanics: insights into a twin DNA polymerase machine.</a> Trends Microbiol. 15, 156-156 (2007).</li><li>Pomerantz, R. T., O'Donnell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+replisome+uses+mRNA+as+a+primer+after+colliding+with+RNA+polymerase.">The replisome uses mRNA as a primer after colliding with RNA polymerase.</a> Nature. 456, 762-762 (2008).</li><li>Heller, R. C., Marians, K. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twin+DNA+pumps+of+a+hexameric+helicase+provide+power+to+simultaneously+melt+two+duplexes.">Twin DNA pumps of a hexameric helicase provide power to simultaneously melt two duplexes.</a> Cell. 109, 757-757 (2002).</li><li>Hanawalt, P. C., Spivak, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=coli+Transcription+repair+coupling+factor+(Mfd+protein)+rescues+arrested+complexes+by+promoting+forward+translocation.">coli Transcription repair coupling factor (Mfd protein) rescues arrested complexes by promoting forward translocation.</a> Nat Rev Mol Cell Biol. 9, 958-958 (2008).</li><li>Hanawalt, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription-coupled+DNA+repair:+two+decades+of+progress+and+surprises.">Transcription-coupled DNA repair: two decades of progress and surprises.</a> Science. 266, 1957-1957 (1994).</li><li>Selby, C. P., Sancar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription-coupled+repair+and+human+disease.">Transcription-coupled repair and human disease.</a> J Biol Chem. 270, 4882-4882 (1995).</li><li>Liu, B., Alberts, B. 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Das Waschen des gestoppt Dehnung Komplex gebunden an die Perlen mit hohem Salz ist, um jede instabile oder nicht-spezifisch gebundene RNAP Molekülen zu entfernen kritisch. RNAP hat eine extrem hohe Affinität für die Primer-Template-Typ von DNA-Strukturen. So ist es notwendig, solche unspezifischen RNAP-DNA-Komplexe zu entfernen, um die Replikation von der Primer-Template durchführen. Konzentration des radioaktiv markierten DNA-Produkte mit dem Qiagen PCR Purification Kit ist ebenfalls entscheidend, um eine ausreichend hohe radioaktive Signal, dass im Gel beobachtet werden zu erhalten.

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<td>Dynabeads M-270</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
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<td>SapI</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>R0569S</td>
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<td>T4 Quick Ligase</td>
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<td>NEB</td>
<td>M2200S</td>
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<td>PCR Purification Kit</td>
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<td>Qiagen</td>
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direct restart of a replication fork stalled by a head-on rna polymerase

In-vivo-Studien deuten darauf hin, dass Replikationsgabeln durch Begegnungen mit Kopf-auf die Transkription Komplexe verhaftet. Doch nach dem Schicksal der replisome und RNA-Polymerase (RNAP) eine Frontalkollision ist unbekannt. Hier finden wir, dass die E. coli replisome Stände beim Zusammenstoß mit einem Kopf-an-Transkription komplex, aber anstelle von zusammenbricht, bleibt das Replikationsgabel sehr stabil und schließlich wieder Dehnung nach Verschieben der RNAP von DNA. Wir finden auch, dass der Transkriptions-Reparatur Kopplungsfaktor, MFD, direkten Neustart der Gabel nach der Kollision durch die Erleichterung Verschiebung der RNAP fördert. Diese Ergebnisse zeigen die innere Stabilität der Replikation Apparat und eine neue Rolle für die Transkription gekoppelte Reparatur-Weg bei der Förderung der Replikation an einem RNAP zu blockieren.

Watch this Scientific Journal Video about Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase at JoVE.com

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Das Schicksal der replisome nach einer Kollision mit einem Kopf-an-RNA-Polymerase (RNAP) ist unbekannt. Wir finden, dass die replisome Stände beim Zusammenstoß mit einem Kopf-an RNAP, sondern nimmt Dehnung nach Verschieben der RNAP von DNA. Mfd fördert die Replikation starten durch Erleichterung der Verschiebung der RNAP nach der Kollision.

Direkte Starten eines Replication Fork von einem Head-On RNA Polymerase Stalled

In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA ...

direct-restart-replication-fork-stalled-head-on-rna

Research

JoVE Journal

Biology

13.5K Aufrufe.  Rockefeller University. Das Schicksal der replisome nach einer Kollision mit einem Kopf-an-RNA-Polymerase (RNAP) ist unbekannt. Wir finden, dass die replisome Stände beim Zusammenstoß mit einem Kopf-an RNAP, sondern nimmt Dehnung nach Verschieben der RNAP von DNA. Mfd fördert die Replikation starten durch Erleichterung der Verschiebung der RNAP nach der Kollision.

Serie: Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Produktion von replikationsdefekte Retrovirus durch transiente Transfektion von 293T-Zellen

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

Forschung

Biologie

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualizing Einzel-Molekül-DNA-Replikation mit Fluoreszenz-Mikroskopie

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

Direkte Beobachtung von Enzymen Replizieren DNA Mit einem Single-Molekül DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Visualizing RNA Localization in Xenopus Oozyten

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

Direkte Lieferung von MIF Morpholinos Into the Zebrafisch Otocyst durch Injektion und Elektroporation Beeinflusst Inner Ear Entwicklung

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro Transkription und Capping von Gaussia-Luciferase mRNA von HeLa-Zelle Transfektion Gefolgt

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; In situ</em&gt; Hybridisierung in whole mount Embryonen und Histologie für Marine Elasmobranchs Angepasst

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

RNA-Isolierung aus Maus Pankreas: Eine Ribonuklease-reichen Gewebe

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

Entwicklung eines Quantitative Rekombinase Polymerase Amplification Assay mit einem internen positiven Kontrolle

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.